Centricon plus 70 filter device

The probiotic strain EcN (serotype O6:K5:H1) was provided by Ardeypharm (GmbH, Herdecke, Germany). OMVs were isolated from culture supernatants of EcN grown overnight at 37°C in Luria-Bertani broth (LB) as previously described (Aguilera et al., 2014 (link)). Briefly, bacterial cells were pelleted by centrifugation at 10,000 × g for 20 min at 4°C. The supernatant was filtered through a 0.22 μm-pore-size filter (Millipore) to remove residual bacteria, concentrated by centrifugation in a Centricon Plus-70 filter device (Millipore) followed by an additional filtration step. OMVs were then recovered by centrifugation at 150,000 × g for 1 h at 4°C, washed and resuspended in sterile PBS. Aliquots were stored at -20°C. The sterility of samples was assessed on LB-agar plates. Protein concentration was measured using the method of (Lowry et al., 1951 (link)).

Membrane Vesicles (MVs) were isolated from culture supernatants following previously described methodology [36 (link)]. Bacterial cells were grown in MMY cultures with the pertinent antibiotics until an OD₆₀₀ of 0.6–0.8 was reached, then pelleted by centrifugation at 10,000× g for 30 min at 4 °C. The supernatants were filtered through a 0.45 μm-pore-size filter (Millipore) and concentrated by centrifugation through a 100 KDa Centricon^® Plus-70 filter device (Millipore). The MVs were collected by ultracentrifugation at 100,000× g for 2 h at 4 °C, washed with phosphate buffered saline (PBS) to eliminate excess polysaccharide and again pelleted at 100,000× g for 2 h at 4 °C. The pellet containing the MVs was resuspended in an appropriate volume of PBS and stored at −20 °C.
MVs were quantified by lipid content, which was determined using the lipophilic fluorescent dye FM4-64 (Thermofisher, Waltham, MA, USA) as previously described [37 (link)]. A fraction of PBS resuspended MVs was incubated with FM4-64 (final concentration of 5 μg/mL in PBS) for 5 min in darkness at room temperature. Vesicles alone and the FM4-64 probe alone were used as negative controls. After excitation at 515 nm, emission at 635 nm was measured with the multiplate reader Varioskan TM LUX (ThermoScientific, Waltham, MA, USA). Fluorescence was normalized for the culture volume from which MVs were isolated.

Bacterial lysates were prepared from cells grown in LB medium. Bacteria were collected by centrifugation (5,000 × g, 10 min, 4°C) and resuspended in phosphate buffer saline (PBS) for sonic disruption on ice. Cell debris was removed by centrifugation at 16,000 × g for 30 min at 4°C and the supernatant filtered through a 0.22 μm-pore-size filter to remove any residual cell.
Outer membrane vesicles were isolated from culture supernatants as described previously (Aguilera et al., 2014 (link)). In brief, bacterial cells grown overnight in LB were pelleted by centrifugation (10,000 × g, 20 min, 4°C); the supernatants were filtered through a 0.22 μm-pore-size filter (Millipore), concentrated by centrifugation in a Centricon Plus-70 filter device (Millipore) followed by an additional filtration step. Vesicles were then collected by centrifugation at 150,000 × g for 1 h at 4°C, washed and resuspended in PBS. Isolated OMVs were examined by transmission electron microscopy after negative staining as described previously (Egea et al., 2007 (link)).
Samples, either lysates or OMVs, were stored at -20°C until use. Sterility of samples was assessed on LB plates. Protein concentration was determined by the method of Lowry et al. (1951) (link).